The crucial role of cyclic AMP in cell proliferation and differentiation has been well-described. The addition of retinoic acid (RA), cholera toxin (CT), or 8-Br cyclic AMP (8-BrcAMP) to a human promyelocytic leukemia cell line (HL60) in culture results in a marked differentiation of the cells to a mature myeloid phenotype, while the addition of the phorbol diester 12-0-tetradecanoyl phorbol-13-acetate (TPA) results in the differentiation of the cells into macrophages. We have detected cAMP-dependent and -independent altered phosphorylations of specific endogenous proteins during RA-\and TPA-induced differentiation of HL60 cells which appear to be specific for each pathway of differentiation. We have noted marked changes in the cAMP-dependent and -independent protein kinase activities which are different in cells differentiated along the macrophage pathway from those differentiated to the mature myeloid phenotype. Retinoic acid and agents raising intracellular cyclic AMP synergistically induced myeloid differentiation. We have recently demonstrated that the addition of low levels of dimethyl formamide, retinoic acid, actinomycin D or hypoxanthine prior to the addition of 8-bromo-cyclic adenosine 3[unreadable]1[unreadable]-5[unreadable]1[unreadable], monophosphate, cholera toxin or the phosphodisterase inhibitor isobutylmethyl-xanthine, results in marked potentiation of differentiation of both HL60 and the RDFD-leukemic cells as manifested by the acquisition of the OKM-1 antigen, the ability to reduce nitroblue tetrazolium or expression of the chemotactic receptor. These results further suggest a role for cAMP in myeloid differentiation. No significant modulations of protein phosphorylations have been detected in the cytosolic fraction during HL60 exposure to RA or AA and cyclic AMP elevating agents. We will investigate modulation of cAMP-dependent and -independent protein phosphorylations in plasma membrane fractions and determine if these phosphorylations are also noted during in vivo phosphorylation utilizing [unreadable]32[unreadable]PO[unreadable]4[unreadable]. In addition, blast forms have been isolated from patients with acute myelogenous leukemia, acute myelomonocytic leukemia, chronic myelomonocytic leukemia and acute lymphocytic leukemia. These cells will be exposed to RA for various periods of time and in vivo protein phosphorylation determined. It is hoped that understanding of this biochemical process of differentiation will allow us to improve current clinical treatments. (M)